Biochemical Molecular Neurobiology Laboratory, Department of Molecular Biology, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314, USA.
The fragile X mental retardation protein (FMRP) is an RNA binding protein that is methylated by an endogenous methyltransferase in rabbit reticulocyte lysates. We mapped the region of methylation to the C-terminal arginine-glycine-rich residues encoded by FMR1 exon 15. We additionally demonstrated that mutation of R(544) to K reduced the endogenous methylation by more than 80%, while a comparable mutant R(546)-K reduced the endogenous methylation by 20%. These mutations had no effect on the subcellular distribution of FMRP, recapitulating previous results using the methyltransferase inhibitor adenosine-2',3'-dialdehyde. Using purified recombinant protein arginine methyltransferases (PRMTs), we showed that the C-terminal domain could be methylated by PRMT1, PRMT3, and PRMT4 in vitro and that both the R(544)-K mutant and the R(546)-K mutant were refractory toward these enzymes. We also report that truncating the N-terminal 12 residues encoded by FMR1 exon 15, which occurs naturally via alternative splicing, had no effect on FMRP methylation, demonstrating conclusively that phosphorylation of serine residue 500 (S(500)), one of the 12 residues, was not required for methylation. Nevertheless, truncating 13 additional amino acids, as occurs in the smallest alternatively spliced variant of FMR1 exon 15, reduced methylation by more than 85%. This suggests that differential expression and methylation of the FMRP exon 15 variants may be an important means of regulating target mRNA translation, which is consonant with recently demonstrated functional effects mediated by inhibiting FMRP methylation in cultured cells.
"A coarse-grained elastic network model  only takes the Cα atom for each amino acid residue into account as “nodes” in a protein network . For each nucleotide, 2 or 3 representative atoms are assigned as nodes [63, 64]. "
[Show abstract][Hide abstract] ABSTRACT: Programmed ribosomal frameshifting (PRF) serves as an intrinsic translational regulation mechanism employed by some viruses to control the ratio between structural and enzymatic proteins. Most viral mRNAs which use PRF adapt an H-type pseudoknot to stimulate -1 PRF. The relationship between the thermodynamic stability and the frameshifting efficiency of pseudoknots has not been fully understood. Recently, single-molecule force spectroscopy has revealed that the frequency of -1 PRF correlates with the unwinding forces required for disrupting pseudoknots, and that some of the unwinding work dissipates irreversibly due to the torsional restraint of pseudoknots. Complementary to single-molecule techniques, computational modeling provides insights into global motions of the ribosome, whose structural transitions during frameshifting have not yet been elucidated in atomic detail. Taken together, recent advances in biophysical tools may help to develop antiviral therapies that target the ubiquitous -1 PRF mechanism among viruses.
Computational and Mathematical Methods in Medicine 04/2012; 2012(17):569870. DOI:10.1155/2012/569870 · 0.77 Impact Factor
"The location of the methylation site required for binding of Tdrkh to MIWI has been identified (Chen et al., PNAS 2009), but it is unclear how Tudor proteins identify binding sites in other proteins, especially those with multiple Tudor domains. Although the sites of methylation for FMRP have been identified (Stetler et al. 2006) and PRMT1 has been identified as a PRMT capable of methylating the protein in cells (Blackwell et al. 2010), additional PRMTs have been shown to methylate FMRP in vitro (Dolzhanskaya et al. 2006) and might also be capable of methylating one or more sites within FMRP in vivo. Methylation of FMRP has been shown to affect RNA association (Blackwell et al. 2010), and the RGG box is required for association with the RNA-binding protein Yb1/p50 (Blackwell and Ceman 2011), suggesting that methylation may also affect protein interactions. "
[Show abstract][Hide abstract] ABSTRACT: Arginine methylation is a post-translational modification that regulates protein function. RNA-binding proteins are an important class of cell-function mediators, some of which are methylated on arginine. Early studies of RNA-binding proteins and arginine methylation are briefly introduced, and the enzymes that mediate this post-translational modification are described. We review the most common RNA-binding domains and briefly discuss how they associate with RNAs. We address the following groups of RNA-binding proteins: hnRNP, Sm, Piwi, Vasa, FMRP, and HuD. hnRNPs were the first RNA-binding proteins found to be methylated on arginine. The Sm proteins function in RNA processing and germ cell specification. The Piwi proteins are largely germ cell specific and are also required for germ cell production, as is Vasa. FMRP participates in germ cell formation in Drosophila, but is more widely known for its neuronal function. Similarly, HuD plays a role in nervous system development and function. We review the effects of arginine methylation on the function of each protein, then conclude by addressing remaining questions and future directions of arginine methylation as an important and emerging area of regulation.
Molecular Reproduction and Development 03/2012; 79(3):163-75. DOI:10.1002/mrd.22024 · 2.53 Impact Factor
"First, glycine-flanked arginine residues within RGG repeat motifs serve as target sites for Type I protein arginine methyltransferases (PRMTs), and methylation of specific arginine residues can have varied effects on a protein's RNA-Binding activity, its ability to interact with other proteins and its intracellular localization [22, 23]. Second, alternative splicing, in and around RG-rich domains has been shown to modulate both nucleic acid binding [24, 25] and protein methylation . "
[Show abstract][Hide abstract] ABSTRACT: Stress granules contain a large number of post-translationally modified proteins, and studies have shown that these modifications serve as recruitment tags for specific proteins and even control the assembly and disassembly of the granules themselves. Work originating from our laboratory has focused on the role protein methylation plays in stress granule composition and function. We have demonstrated that both asymmetrically and symmetrically dimethylated proteins are core constituents of stress granules, and we have endeavored to understand when and how this occurs. Here we seek to integrate this data into a framework consisting of the currently known post-translational modifications affecting stress granules to produce a model of stress granule dynamics that, in turn, may serve as a benchmark for understanding and predicting how post-translational modifications regulate other granule types.
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